The present invention relates to transgenic non-human animals wherein the mature form of at least one lymphocytic cell type is substantially depleted. More particularly, the invention relates to transgenic mice wherein mature T cells or plasma cells are depleted.
The immune response is a complex defense system that is able to recognize and kill invading organisms such as bacteria, viruses, fungi and possibly also some types of tumor cells. The most characteristic aspects of the immune system are the specific recognition of antigens, the ability to discriminate between self and non-self antigens and a memory-like potential that enables a fast and specific reaction to previously encountered antigens. The vertebrate immune system reacts to foreign antigens with a cascade of molecular and cellular events that ultimately results in the humoral and cell-mediated immune response.
The major pathway of the immune defense commences with the trapping of the antigen by accessory cells such as dendritic cells or macrophages and subsequent concentration in lymphoid organs. There, the accessory cells present the antigen on their cell surface to subclasses of T cells classified as mature T helper cells. Upon specific recognition of the processed antigen the mature T helper cells can be triggered to become activated T helper cells. The activated T helper cells regulate both the humoral immune response by inducing the differentiation of mature B cells to antibody producing plasma cells and control the cell-mediated immune response by activation of mature cytotoxic T cells.
Naturally occurring processes sometimes result in the modulation of immune system cell types. Acquired immunodeficiency syndrome (AIDS) is a devastating infectious disease of the adult immune system which significantly affects cell-mediated immunity. This disease is manifested by profound lymphopenia which appears to be the result of a loss of T-lymphocytes which have the helper/inducer phenotype T4 as defined by the monoclonal antibody OKT4 (Fauci, A., et al. (1984) Annals. Int. Med. 100, 92). Other clinical manifestations include opportunistic infections, predominantly Pneumocystis carinii pneumonia, and Karposi""s sarcoma. Other disease states include Severe Combined Immuno Deficiency Syndrome (SCID) wherein T, B or both cell types may be depleted in humans. It is the existence of diseases affecting the immune system, such as AIDS and SCID, which has created the need for animal model systems to study the epitology and potential treatment of such disease states.
T lymphocytes recognize antigen in the context of self Major Histocompatibility Complex (MHC) molecules by means of the T cell receptor (TCR) expressed on their cell surface The TCR is a disulfide linked heterodimer noncovalently associated with the CD3 complex (Allison, J. P., et al. (1987) Ann. Rev. Immunol. 5, 503). Most T cells carry TCRs consisting of xcex1 and xcex2 glycoproteins. T cells use mechanisms to generate diversity in their receptor molecules similar to those operating in B cells (Kronenberg, M., et al. (1986) Ann. Rev. Immunol. 4, 529; Tonegawa. S. (1983) Nature 302, 575). Like the immunoglobulin (Ig) genes, the TCR genes are composed of segments which rearrange during T cell development. TCR and Ig polypeptides consist of amino terminal variable and carboxy terminal constant regions. The variable region is responsible for the specific recognition of antigen, whereas the C region functions in membrane anchoring and in transmitting of the signal that the receptor is occupied, from the outside to the inside of the cell. The variable region of the Ig heavy chain and the TCR xcex2 chain is encoded by three gene segments, the variable (V), diversity (D) and joining (J) segments. The Ig light chain and the TCR xcex1 chain contain variable regions encoded by V and J segments only.
The V, D and J segments are present in multiple copies in germline DNA. The diversity in the variable region is generated by random joining of one member of each segment family. Fusion of gene segments is accompanied by insertion of several nucleotides. This N-region insertion largely contributes to the diversity, particularly of the TCR variable regions (Davis and Bjorkman (1986) Nature 334, 395), but also implies that variable gene segments are often not functionally rearranged. The rearrangement of gene segments generallyoccurs at both alleles. However, T and B cells express only one TCR or Ig respectively and two functionally rearranged genes within one cell have never been found. This phenomenon is known as allelic exclusion.
During B development the rearrangement process starts at both heavy chain gene alleles. First a D segment is fused to a J segment followed by ligation of a V segment of the DJ join. If the VDJ joining is productive, further rearrangement of the other heavy chain allele is blocked, whereas rearrangement of the light chain loci is induced (Reth, M., et al. (1985) Nature 317, 353).
In both B and T cells, partially (DJ) and completely (VDJ) rearranged genes reportedly are transcribed giving rise to two differently sized RNA molecules (Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339; Born, W., et al. (1987) TIG 3, 132). In B cells the DJ transcripts can be translated into a Dxcexc-chain, a truncated form of the Igxcexc heavy chain that lacks a V segment derived sequence. In general, that Dxcexc-chain is present in minor amounts, if at all. However, in one subclone (P4-11) of the 300-19 cell line, a transformed pre-B cell line which differentiates in vitro to Ig producing B cells, the expression of the Dxcexc-chain is reportedly very high (Reth, M., et al. (1985) Nature 317, 353). This reference also reports that the heavy chain gene alleles in the P4-11 clone are blocked at the DJ rearrangement stage in cell culture and that such cells show a very high frequency of light chain gene rearrangements. This has led to the suggestion that the Dxcexc protein contains some of the regulatory determinales necessary for gene assembly (Yancopoulos, G., et al. (1986) Ann. Rev. Immunol. 4, 339, 356).
Transgenic mice containing functionally rearranged Ig genes reportedly have been used in studying several aspects of Ig gene expression, e.g. tissue specific expression, the mechanism of segment rearrangement, allelic exclusion and repertoire development (Storb, U. (1987) Ann. Rev. Immunol. 5, 151). It has also been reported that the transgenic heavy chain polypeptide only inhibits the complete VDJ rearrangement of endogenous heavy chain genes if it contains a transmembrane domain (Storb. 1987; Iglesias, A., et al. (1987) Nature 330, 482; Nussenzweig, M., et al. (1987) Science 236, 816; Nussenzweig, M., et al. (1988) J. Exp. Med. 167, 1969).
Recently, the inventors reported that functionally rearranged TCRxcex2 genes can be appropriately expressed in transgenic mice (Krimpenfort, P., et al. (1988) EMBO 7, 745). This functional TCRxcex2 chain gene prevents expression of endogenous xcex2 genes by inhibiting complete VDJ joining (Uematsu, Y., et al. (1988) Cell 52, 831).
Two different types of T cells are involved in antigen recognition within the self MHC context. Mature T helper cells (CD4+CD8xe2x88x92) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (CD4xe2x88x92CD8+) recognize antigen in the context of class IMHC determinants (Swain, S. L. (1983) Immun. Rev. 74, 129-142; Dialynas, P. D., et al. (1983) Immun. Rev. 74, 29-56). It has been reported that class II- specific CD4+CD8xe2x88x92 helper T cells (also referred to as T4 cells) fail to develop in mice neonatally treated with anti-class II MHC monoclonal antibody (Kruisbeek, A. M., et al. (1983) J. Exp. Med. 157, 1932-1946; Kruisbeek, A. M., et al. (1985) J. Exp. Med. 161, 1029-1047). Similarly, it has recently been reported that mice chronically treated with anti-class I MHC monoclonal antibody from birth have a significantly reduced population of CD4xe2x88x92CD8+ cells and cytotoxic T cell precursors (Marusic-Galesic, S., et al. (1988) Nature 333, 180-183). Although selected T cell populations apparently can be produced by such methods, continuous administration of antibody is required which often results in adverse side effects in such mice.
A different strategy to deplete specific cell lines has recently been identified wherein specific cell destruction is induced by administration of a toxic metabilite. Specifically, transgenic mice reportedly were produced containing a Herpes Simplex Virus Thymidine Kinase (HSV-TK) transgene fused to the Ig promoter/enhancer. Transgenic cells that express the HSV-TK are not affected. However, upon administration of a nucleoside analog that can be phosphorylated by the transgenic HSV-TK gene, replicating cells expressing the HSV-TK gene are killed (Heyman, et al. (1988) UCLA Symposia on Molecular and Cellular Biology, 73, 199.
Another approach to depletion of specific cell types has been reported using tissue specific expression of a bacterial toxin. Specifically, mice carrying an elastase/diptheria toxin A (DT-A) fusion gene lacked a normal pancreas (Palmeter, et al. (1987) Cell 50, 435). In addition, it has been reported that microphtalmia in transgenic mice resulted from the introduction of the DT-A gene fused to the xcex12-crystallin promoter (Bretman, et al. (1987) Science 238, 1563).
Transgenic mice reportedly have also been constructed that express an xcex1xcex2TCR in a large fraction in their T cells which is specific for a minor histocompatibility antigen (H-Y) present on male, but not female, cells (Kisielow, P., et al. (1988) Nature 333, 742-746). This very recent reference reports that cells containing the TCR for the H-Y antigen were frequent in female but not in male transgenic offspring. The xcex1xcex2 TCR in such transgenic mice apparently contains all the segments and regions required for a functional TCR.
The reference discussed above are provided solely for the disclosure prior to the filing date of the present application and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosures by virtue of prior invention.
Given the state of the art, it is apparent that a need exists for animal model systems to study diseases which effect the immune system including infectious diseases such as AIDS. Accordingly, it is an object herein to provide transgenic non-human animals and methods for making the same which have a phenotype characterized by the substantial depletion of a mature lymphocytic cell type otherwise naturally occurring in the species from which the transgenic is derived.
It is also an object herein to provide transgenic non-human animals substantially depleted in mature T cells or plasma cells.
It is a further object herein to provide transgenic mice substantially depleted in mature T cells or plasma cells.
Still further, it is an object herein to provide transgenes capable of producing such transgenic non-human animals.
Further, it is an object herein to provide methods for producing transgenic non-human animal having at least one of the above identified phenotypes.
The invention is based on the discovery that transgenic non-human animals depleted in a lymphatic cell type can be produced by disrupting the expression of a functional lymphocytic polypeptide required for maturation of the lymphocytic cell type. This lymphocytic polypeptide is otherwise expressed by the non-human animal from which the transgenic animal is derived.
In one aspect, the invention provides transgenic non-human animals having a phenotype characterized by the substantial depletion of a mature lymphocytic cell type otherwise naturally occurring in the species from which the transgenic animal is derived. This phenotype is conferred in the transgenic animal by a transgene contained in at least the precursor stem cell of the lymphocytic cell type which is depleted. The transgene comprises a DNA sequence encoding a lymphatic polypeptide variant which inhibits formation of the depleted lymphocytic cell type. Generally, such inhibition occurs when the lymphatic polypeptide variant is expressed in a precursor to the lymphocytic cell type.
In those cases where the lymphatic polypeptide variant is expressed, the variant is believed to be capable of suppressing expression of at least one set of cognate endogenous alleles normally expressed during differentiation of the precursor stem cell to the mature lymphocytic cell type. The lymphatic polypeptide variant, however, lacks a functional domain necessary for maturation of the lymphocytic cell type which would otherwise be provided by either or both of the suppressed endogenous alleles.
Within the context of transgenic animals deficient in T cells, the transgene encodes a lymphatic polypeptide variant comprising a portion of a TCRxcex2 chain. The transgene encoding the TCRxcex2 variant chain typically retains sequences encoding at least the transmembrane sequence found in the C region of a naturally occurring TCRxcex2 chain. This sequence may be operably linked to an appropriate signal sequence. It lacks, however, sequences encoding all or part of the variable region. The C region contained by such a lymphatic polypeptide variant is capable of suppressing the expression of endogenous TCR alleles thereby preventing the membrane expression of functional heterodimeric TCRs. Normal T cell maturation is thereby abrogated.
In the case of non-human transgenic animals substantially depleted in antibody secreting plasma cells, the transgene similarly encodes a lymphatic polypeptide variant containing at least the transmembrane sequence of the C region of the Ig heavy chain. A signal sequence may also be operably linked to the transgene encoding the lymphatic polypeptide variant.
The invention also includes transgenes comprising a DNA sequence encoding a lymphatic polypeptide variant.
Further, the invention includes a method for producing a transgenic non-human animal substantially depleted in a mature lymphocytic cell type. The method comprises introducing a transgene into an embryonal target cell. The transgene encodes a lymphatic polypeptide variant and is capable of inhibiting formation of a mature lymphocytic cell type. The thus transformed transgenic embryonal target cell is thereafter transplanted into a recipient female parent from which offspring having the desired phenotype are identified.